Systematic in vitro assessment of responses of roGFP2-based probes to physiologically relevant oxidant species

The genetically encoded probes roGFP2-Orp1 and Grx1-roGFP2 have been designed to be selectively oxidized by hydrogen peroxide (H2O2) and glutathione disulfide (GSSG), respectively. Both probes have demonstrated such selectivity in a broad variety of systems and conditions. In this study, we systematically compared the in vitro response of roGFP2, roGFP2-Orp1 and Grx1-roGFP2 to increasing amounts of various oxidant species that may also occur in biological settings. We conclude that the previously established oxidant selectivity is highly robust and likely to be maintained under most physiological conditions. Yet, we also find that hypochlorous acid, known to be produced in the phagocyte respiratory burst, can lead to non-selective oxidation of roGFP2-based probes at concentrations ≥2µM, in vitro. Further, we confirm that polysulfides trigger direct roGFP2 responses. A side-by-side comparison of all three probes can be used to reveal micromolar amounts of hypochlorous acid or polysulfides. •Comparison of the in vitro response of 3 roGFP2-based probes to a panel of oxidants.•The probes' selectivity is maintained in the absence of highly reactive species.•HOCl and polysulfides lead to unspecific probe oxidation.•Peroxynitrite oxidizes roGFP2-Orp1 with kinetics comparable to H2O2.•Similar responses of probes indicate the presence of highly reactive species.